Contemporary research in the life sciences, including recent advances in genomics and proteomics, has led to the discovery of thousands of proteins with potential diagnostic and/or therapeutic significance. For example, some of these proteins are biological markers for organ activity, disease processes, or drug action. The ability to monitor slight differences in the amounts of these proteins as well as other biological macromolecules, in the smallest possible detection volumes, down to the level of single cells, is of utmost importance not only for proteomics research but also for biomedical diagnostics in general.1a To date, antibody-based immunological assays (e.g., ELISA) are the most commonly used diagnostic methods for protein detection; typically, they are not as sensitive or specific as methods for detection of specific nucleic acid sequences, DNA microarrays for instance.1b Thus, there is a need for DNA-based technologies capable of detecting proteins in very low quantities.
Despite a lack of complete understanding of the mechanistic details of electron transfer through DNA, long-range electron transfer in double-stranded DNA is generally believed to be the result of a multi-step hopping reaction1-2. The consensus view is that a continuous base-stacking throughout the DNA duplex is essential for efficient charge transfer. It has been shown that efficiency of charge transfer is reduced in duplexes containing mismatches3-5 and bulges6. Proteins that bind and disrupt continuous base-stacking in duplex DNA also reduce the efficiency of electron transfer past the site of helix disruption7-8. Despite the importance of a continuous base stack, not all perturbations to the helix prevent charge transfer, as it has been observed in helices containing abasic sites9 and through short, single stranded overhangs10. However, even these latter structures are believed to base-stack to some extent, which permits charge transfer through them.
Detection of charge transfer in DNA has been detected both directly and indirectly. Dehydrated DNA duplexes11 or DNA fibers12a,b positioned between metal electrodes have had their conductivity measured directly. Indirect measurement of DNA conductivity has been made in aqueous solution, after inducement of charge transfer with a photoexcitable moiety (such as anthraquinone13, or rhodium(III) complexes with aromatic ligands14). The photoexcitable moiety is attached to one end of a duplex such that it lies in intimate contact with the π-stack of the DNA base pairs. The photo-excited states of anthraquinone and rhodium(III) complexes are powerful oxidising agents, and are able to collect electrons from guanines (via generation of a mobile radical cation, or electron hole) within the DNA duplex, from reported distances of up to >200 Å away from the ligand15a,b). According to the putative “multi-step hopping” mechanism referred to above, the radical cation moves from guanine to guanine (guanine is the base with the lowest ionization potential). A guanine upon which the mobile radical cation is transiently localized is somewhat susceptible to reaction with water and dissolved oxygen, leading to the formation of oxidation products such as diaminooxazalone and 2-aminoimidazalone16. As described herein, the position of the latter products along a DNA strand can readily be detected by sequencing gel-electrophoresis, since these products are base-labile and cause site-specific strand breakage on being treated with hot piperidine.
Despite disagreements on the precise mode of charge transfer within DNA duplexes, investigators are in agreement that the electrical conductivity of DNA is dependent on its conformational state—specifically, on the integrity of its π-stacking. While much of the research on DNA conduction has focused on “static” or relatively immobile DNA structures, the purpose of the present invention is to exploit changes in the conductivity of DNA, dependent on changes in its conformational state, to provide information about the DNA's environment—such as the presence or absence of a specific analyte. In other words, if conformational change in the DNA results from the binding of a particular analyte, then this should correlate with a change in the DNA's conductivity, providing the basis for an analyte sensing device constructed from DNA or other oligonucleotides.
In nature, DNA is known to bind a variety of small molecule as well as macromolecular ligands. However, recent innovations in in vitro selection (SELEX) methods have resulted in DNA (as well as RNA) “aptamer” sequences, which are capable of specifically binding a variety of molecular species, including many that normally do not interact with DNA or RNA17. Such aptamer oligonucleotides frequently exhibit induced-fit folding behaviour (reviewed by Hermann & Patel18), whereby the aptamer itself, largely unstructured in solution, undergoes significant compaction and structural stabilization upon binding its cognate ligand. Due to the ease with which novel, made-to-order aptamers can be selected from large, random sequence DNA and RNA libraries, and their generally impressive selectivity and affinity, they are widely regarded as ideal recognition elements for biosensor applications.18a 
Barton and colleagues19 have reported the electronic detection of a DNA-binding protein, HhaI methyltransferase, by virtue of the protein's interference in the charge conduction path of a duplex DNA. HhaI methylase works by binding to a target G*CGC site on a double-helix, and extruding the target cytosine base (marked with an asterisk, above) out of the helix in order to methylate it. This extrusion naturally disrupts the conduction path through the helix and, thus, the level of conduction through the helix. While this approach demonstrates protein-modulation of charge-transfer through DNA, it requires the selection of a protein capable of extruding a base out from the DNA helix and is therefore not of general application. For example, unlike the present invention, the method could not be readily extended as a means for the detection of any protein, large or small, DNA-binding or not.
It is also known in the prior art to detect conformational change in duplex DNA by binding of divalent metal ions. Lee and colleagues20 have reported a methodology for the electronic detection of a DNA-binding protein. Following the binding of the protein to its binding site upon a DNA duplex, the DNA is converted to a metal-bound form (“M-DNA”), with a significantly higher conductivity than that of standard B-DNA. The presence of the bound protein, however, interferes with M-DNA formation by its binding site, and therefore affects the overall conductivity of the duplex. While this approach is promising, the efficacy of this method for use in the detection of proteins that do not naturally bind to DNA, or which bind to non-duplex elements of DNA or RNA, has not yet been reported.
The need has therefore arisen for improved biosensors of general application for analyte detection, and in particular, biosensors for rapidly detecting proteins at low concentrations in biological fluids. Since the detection means is electronic, the potential exists for use of such sensors for rapid and automated chip-based detection of small molecules as well as of proteins and other macromolecules. The sensors are also potentially useful as nanoelectronic switches and junction devices simulating solid state electronic logic gates.